Preparing the samples

 

To minimize evaporation, prepare samples immediately before running the Protein Cleanup protocol, or keep the plates lidded until the run begins.

 

When preparing the samples:

•	Remove macromolecular particulates before the samples are loaded onto AssayMAP cartridges.

•	Adjust the buffer composition to the optimal binding conditions (for example, pH).

•	Determine the sample volume to load on the AssayMAP cartridges.

•	Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulate

 

 

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45‑µm filter or centrifuged at a high g‑force before loading on an AssayMAP cartridge.

 

 

 

Return to top

Adjusting the buffer composition

The Protein Cleanup protocol can be used for a wide variety of proteins, but the binding and elution conditions should be determined empirically after consulting the relevant scientific literature.

Antibodies denatured with guanidine HCL should be acidified and diluted to reduce the guanidine HCL concentration before loading onto AssayMAP RP‑W cartridges. The binding capacity of the RP-W cartridges decreases when samples have a pH > 3 and guanidine concentrations > 1.5 M. The magnitude of this affect is likely to be protein dependent and, therefore, should be determined empirically. Acidification can be accomplished with TFA, formic acid or acetic acid.

Avoid samples containing organic solvents or some types of detergents as they might inhibit binding to the cartridge. For example, loading samples in a buffer containing greater than 5% acetonitrile might inhibit binding of some proteins. If you have concerns about a specific buffer component, you should survey the scientific literature for any known effects of this type of molecule on reversed-phase resins.

The Reagent Transfer utility is a simple way to adjust the pH or dilute your sample before loading it onto the RP‑W cartridge. For instructions, see Reagent Transfer v2.1. Alternatively, use the In-Solution Digestion: Single Plate application to perform these tasks, especially when desalting a denatured protein. For instructions, see In‑Solution Digestion: Single Plate v1.2 User Guide.

Note: A reverse-phase protein clean up may not work for all proteins because solubility in the high organic solvent, which is required for elution, can be an issue for some proteins. In addition, when desalting a denatured protein, the removal of the denaturation reagent may cause solubility problems.

 

Return to top

Determining the volume of sample to load

The Protein Cleanup protocol permits loading of up to 1000-µL sample volume onto the AssayMAP RP-W cartridge. If the sample volume is greater than 250 µL, the protocol will iteratively load samples onto the cartridges to stay within the maximum AssayMAP syringe volume (250 µL).

What is the binding capacity of the cartridge?

Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity:

•	Quantitative binding capacity. The maximum mass of protein that can bind to the cartridge in a single pass, where less than 10% of the load appears in the flow-through. See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed quantitative cartridge binding capacity.

The quantitative binding capacity depends on the composition of the solution in which the protein is bound. For denatured antibodies, the binding capacity is slightly reduced when there is greater than 1.5 M guanidine in the sample and if the sample is not acidified.

•

Total binding capacity. The maximum mass of protein that can bind to the cartridge. This can only be achieved only by loading significantly more protein than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity. See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed total binding capacity information for RP-W cartridges.

What is the concentration of the target in the sample?

If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

 

 

Return to top

Preparing the sample plates

Planning the microplate setup

Before transferring the samples, you should plan the layout of the samples in the microplate.

Consider the following:

•	You can process 1 to 96 samples in parallel. The position of the samples in the microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must match the locations of the buffer solutions in microplates and reservoirs.

•	If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the following figure shows.

•

The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based upon the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

•	If the number of samples you have is not a multiple of 8, use the AssayMAP Resin-Free cartridges to fill the empty column and row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash step.

Figure. Example of sample microplate and reservoir layout

 

Transferring the samples to the microplate

 

A small volume excess is required in all labware types to ensure proper volume transfer.

 

An excess (overage) volume ensures that a microplate well or column does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance.

The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. See Using the Reagent Volume Calculator for Protein Cleanup.

Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate:

1	Use the Reagent Transfer utility to adjust the buffer composition of the samples for protein cleanup (that is, to change the pH or denaturant concentration). For instructions, see Reagent Transfer v2.1.

2	Optional. Run the Reagent Transfer utility to move samples into one of the labware options for deck location 4. For instructions, see Reagent Transfer v2.1.

3	If necessary, centrifuge the sample labware to remove bubbles.

 

Return to top